EP2007416B1 - Parathormon (pth) zur verwendung in der behandlung der ischämie - Google Patents

Parathormon (pth) zur verwendung in der behandlung der ischämie Download PDF

Info

Publication number
EP2007416B1
EP2007416B1 EP07724213.9A EP07724213A EP2007416B1 EP 2007416 B1 EP2007416 B1 EP 2007416B1 EP 07724213 A EP07724213 A EP 07724213A EP 2007416 B1 EP2007416 B1 EP 2007416B1
Authority
EP
European Patent Office
Prior art keywords
pth
pharmaceutical composition
cells
fragments
csf
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP07724213.9A
Other languages
English (en)
French (fr)
Other versions
EP2007416A2 (de
Inventor
Wolfgang M. Franz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Maximilians Universitaet Muenchen LMU
Original Assignee
Ludwig Maximilians Universitaet Muenchen LMU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ludwig Maximilians Universitaet Muenchen LMU filed Critical Ludwig Maximilians Universitaet Muenchen LMU
Priority to SI200731997T priority Critical patent/SI2007416T1/en
Priority to PL07724213T priority patent/PL2007416T3/pl
Publication of EP2007416A2 publication Critical patent/EP2007416A2/de
Application granted granted Critical
Publication of EP2007416B1 publication Critical patent/EP2007416B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to pharmaceutical compositions comprising parathyroid hormone (PTH) and fragments thereof, preferably PTH (1-34), for use in the treatment of ischemic heart diseases.
  • the pharmaceutical composition comprising PTH or PTH fragments is used for recruiting stem cells into tissue suffering from ischemia, wherein said stem cells are preferably capable of repairing and/or regenerating said tissue suffering from ischemia.
  • PTH can be used in the pharmaceutical composition alone or in combination with a DPP IV antagonist or with a G-CSF or a G-CSF fragment.
  • hematopoietic stem cells The mobilization of hematopoietic stem cells has become an established method for autologous stem cell transplantation. Under steady-state conditions the number of hematopoietic stem cells is much lower in peripheral blood than in bone marrow. Mobilization of these cells with chemotherapy and/or growth factors made it able to increase the concentration in the peripheral blood, making stem cell transplantation with autologous hematopoietic stem cells from the peripheral blood possible. This method results in a more gentle treatment and a faster hematologic recovery 1 .
  • cytokine-mediated mobilization of hematopoietic stem cells has become an established method in the field of autologous stem cell transplantation from peripheral blood.
  • Parathyroid hormone is well known as one of the two major hormones modulating calcium and phosphate homeostasis. It is responsible for maintaining serum ionized calcium concentrations within a narrow range, through its action to stimulate renal tubular calcium reabsorption and bone resorption. On a more chronic basis, parathyroid hormone also stimulates the conversion of calcidiol to calcitriol in renal tubular cells, thereby stimulating intestinal calcium absorbtion 12-16 . In recent years, the receptor for parathyroid hormone was shown to be expressed in several tissues suggesting more complex functions of this hormone 17,18 .
  • HSC hematopoietic stem cell
  • exogenous PTH(1-34) and its structurally related endogenous secreted peptide PTHrP are potent dilators of the arterial bed 30,31 .
  • PTH (1-34) dose dependently increases regional myocardial blood flow 31,32 .
  • PTH (1-34) administration in a reperfusion model in pigs after myocardial infarction improved myocardial contractility and myocardial perfusion 32 .
  • Arterial vasodilation is based on the activation of PTH/PTHrP receptor type I 33 which is known to be expressed on smooth muscle cells. PTH/PTHrP receptor activation results in an increase of cAMP production.
  • PTH has cardiovascular functions such as vasodilatation, increased myocardial blood flow, hypotensive effects, myocardial hypertrophy, positive chronotropic and contractility effects. The latter effect is, however, controversially discussed in the art. Moreover, it has also been observed in clinical studies that the progress of cardiovascular disease may be associated with PTH; see Wing et al. (1984), Contr. Nephrol. , Behap et al. (1985), Nephron or London et al. (1987), Kidney Int. . Mice lacking the PTH/PTHrP receptor type I die at the midgestational age around day 12 due to abrupt cardiomyocyte necrosis showing the essential role of PTH in the early cardiovascular development. Moreover, PTH(1--34) treatment induced a higher SDF-1 mRNA level in the bone marrow 35 .
  • Parathyroid hormone-related protein is actually a family of protein hormones produced by most if not all tissues in the body. A segment of PTHrP is closely related to parathyroid hormone, and hence its name, but PTHrP peptides have a much broader spectrum of effects. Parathyroid hormone and some of the PTHrP peptides bind to the same receptor, but PTHrP peptides also bind to several other receptors. PTHrP was discovered as a protein secreted by certain tumors that caused hypercalcemia (elevated blood calcium levels) in affected patients.
  • hypercalcemia elevated blood calcium levels
  • Ischemia is a restriction in blood supply, generally due to factors in the blood vessels, with resultant damage or dysfunction of tissue. Ischemia is a feature of heart diseases, transient ischemic attacks, cerebrovascular accidents, ruptured arteriovenous malformations, and peripheral artery occlusive disease. Thus, ischemia affects almost all organs and tissues. Tissues especially sensitive to inadequate blood supply are the heart, the kidneys, and the brain. Ischemia in brain tissue, for example due to stroke or head injury, causes a process called the ischemic cascade to be unleashed, in which proteolytic enzymes, reactive oxygen species, and other harmful chemicals damage and may ultimately kill brain tissue.
  • CAD coronary artery disease
  • MI myocardial infarction
  • PCI primary coronary intervention
  • Ballen et al. (Blood 106 (2005), page 557 A ) report that PTH may improve autologous stem cell mobilization.
  • Calvi et al. (Nature 425 (2003), pages 841-846 ) disclose that osteoblastic cells regulate the hematopoietic stem cell niche.
  • Horowitz et al. J. Clin. Invest. 83 (1989), pages 149-157 ) it is described that PTH and LPS induce minimal osteoblast-like cells.
  • WO 2004/056186 A1 refers to cell-based VEGF-delivery.
  • Orlic et al. (PNAS 98 (2001), pages 10344-10349 ) report how mobilized bone marrow cells repair the infected heart.
  • the present invention provides remedies for ischemia, in particular, means, methods and uses for preventing and/or treating ischemia.
  • the present invention relates to the use of parathyroid hormone (PTH) or specific fragments thereof for the preparation of a pharmaceutical composition for recruiting stem cells into tissue suffering from ischemia and/or apoptosis.
  • Said stem cells are upon administration of PTH or specific fragments thereof believed - without being bound by theory - to be mobilized from the bone marrow so as to be present in the periphery. It is, moreover, assumed that said stem cells are preferably recruited into tissue suffering from ischemia.
  • PTH or specific fragments thereof has a direct beneficial effect on cells of tissue suffering from ischemia through binding to the PTH receptor respectively. Accordingly, it is believed that PTH or specific fragments thereof has a beneficial effect on tissue suffering from ischemia due to the effect on stem cells as described herein and/or due to a direct effect on cells of tissue suffering from ischemia.
  • the present invention is based on the surprising finding that stem cells can be recruited from the bone marrow into the periphery, preferably into tissue suffering from ischemia by parathyroid hormone (PTH) or specific fragments thereof. Accordingly, the present inventors aimed to define in a murine model system the impact of PTH on stem cells mobilization, vessel growth, post myocardial infarction survival as well as functional parameters of infarcted myocardium 6 days and 30 days after a surgical procedure further detailed herein below and in the appended Example.
  • the data obtained in the murine model with respect to the prevenetion and/or treatment of ischemia of the heart are generalizable for the prevention and/or treatment of ischemia of other tissues and/or organs as described herein.
  • SDF-1 (CXCL12) is - among others - a substrate for CD26, which is a membrane bound, extracellular dipeptidyl peptidase that splices proteins at their N-terminal end after the aminoacid-sequence X-alanine or X-proline.
  • CD26 A large number of bone marrow stem cells express CD26 on the cell surface.
  • DPP IV is the abbreviation for dipeptidylpeptidase IV and is also referred to as CD26 herein and also in the literature.
  • Dipeptidyl peptidase IV (DPP-IV or CD26) cleaves dipeptides N-terminal after X-Pro/X-Ala and degrades for example the substrates SDF-1 and GLP-1.
  • Dipeptidyl peptidase IV (DPP-IV or CD26) can be inhibited, for example, by diprotin A.
  • DPP IV antagonist also interchangeably used herein as "DPP IV inhibitor”
  • PTH parathyroid hormone
  • DPP IV antagonists are known in the art, e.g., Diprotin A, Vildagliptin or Sitagliptin.
  • DPP IV antagonists are also described, for example, in US2003/0119750 , WO 2005/063750 , DE-A1 1 010 0053 , US 2004/147434 , WO 2003/002596 , WO 2007/035665 , EP 1 743 655 , US 2006/270701 , US 2006/217428 , WO 2005/025554 , AU 2003261487 , CA 2 471 204
  • DPP-IV inhibitors like Vildagliptin or Sitagliptin are already approved for clinical use in diabetes mellitus and are thus within the scope of the present invention.
  • inhibitor defines in the context of the present invention a compound or a plurality of compounds which interact(s) with DPP-IV such that the cleavage of dipeptides N-terminal after X-Pro/X-Ala is reduced and/or the degradation of, for example, the substrates SDF-1 and GLP-1 is reduced.
  • plurality of compounds is to be understood as a plurality of substances which may or may not be identical. The plurality of compounds may preferably act additively or synergistically.
  • Said compound or plurality of compounds may be chemically synthesized or microbiologically produced and/or comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms.
  • said compound(s) may be known in the art but hitherto not known to be capable of reducing the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 mediated by DPP- IV.
  • the interaction of the inhibitor with DPP-IV such that the cleavage of dipeptides N- terminal after X-Pro/X-Ala is reduced and/or the degradation of, for example, the substrates SDF-1 and GLP-1 is reduced can, in accordance with this invention e.g.
  • DPP-IV be effected by a reduction of the amount of the DPP-IV in cells, in tissues comprising said cells or subjects comprising said tissues or cells for example by aptamers, antisense oligonucleotides, iRNA or siRNA which specifically bind to the nucleotides sequences encoding said DPP-IV or by ribozmes which specifically degrade polynucleotides which encode DPP-IV; by blocking the binding site of DPP-IV for its substrates; by competitive or allosteric inhibition of the cleavage of dipeptides N- terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 or by otherwise reducing or preventing the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 , for example by directing antibodies and/or aptamers to DPP-IV and thereby reducing
  • an example of an inhibitor of this invention is an antibody, preferably an antibody the binding of which interferes with the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 mediated by DPP-IV; an antisense construct, iRNA, siRNA or ribozyme constructs directed against a transcript or the coding nucleotide sequence of DPP-IV; nucleotide sequences encoding such constructs and compounds which inhibit the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1.
  • reduced or “reducing” as used herein defines the reduction of the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1
  • inhibitors that act in a reversible manner and do not block biochemical processes completely, because such drugs can be applied in a dosage that complies with the desired effect.
  • the inhibitor at least reduces the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 as mediated by DPP-IV to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% when compared to the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 that is achieved without the addition of said inhibitor.
  • the reduction will also depend on the dosage and on the way of administration of the inhibitor.
  • the dosage regimen utilizing the inhibitor of the present invention is therefore selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; and the particular compound employed. It will be acknowledged that an ordinarily skilled physician or veterinarian can easily determine and prescribe the effective amount of the compound required to prevent, counter or arrest the progress of the condition.
  • Said inhibitor(s) specifically reduce(s) the cleavage of dipeptides N-terminal after X-Pro/X- Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 mediated by DPP-IV.
  • the term "specifically reduce(s)" used in accordance with the present invention means that the inhibitor specifically causes a reduction of the cleavage of dipeptides N-terminal after X-Pro/X-Ala and/or the degradation of, for example, the substrates SDF-1 and GLP-1 mediated by DPP-IV but has no or essentially has no significant effect on other cellular proteins or enzymes.
  • DPP IV antagonist or "DPP IV inhibitor” when used herein also encompasses an agent or a drug or a compound that inhibits or antagonizes the physiological effect of DPP IV. It also encompasses competitive, non-competitive, functional, non-functional and chemical antagonists which inhibit or antagnoize the physiological effect of DPP IV.
  • Any compound can be tested by methods known in the art for its effect on DPP IV, i.e. whether or not it can act as a DPP IV inhibitor.
  • a continuous fluorometric assay can be employed using, e.g. the peptide Gly-Pro-AMC, which is cleaved by the enzyme to release the fluorescent aminomethylcoumarin (AMC).
  • a typical reaction contained 50 pmol/l enzyme, 50 ⁇ mol/l Gly-Pro-AMC, and buffer (100 mmol/l HEPES, pH 7.5, 0.1 mg/ml BSA) in a total reaction volume of 100 ⁇ l.
  • Liberation of AMC can be monitored using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
  • the enzyme used in these studies can be soluble, bound or immobilized and is preferably a human protein produced, for example, in a baculovirus expression system. Accordingly, in order to measure a DPP IV inhibiting activity of a compound, such a compound is added to the above described assay and the skilled person can easily monitor whether AMC release is inhibited in comparison to a test reaction in which the compound to be tested is not added.
  • parathyroid hormone which is known as a regulating hormone in calcium and phosphate homeostasis, is an effective agent to mobilize cells from bone marrow so as to be present in the periphery from where said stem cells are - without being bound by theory - due to the action of PTH preferably recruited into ischemic tissues and/or organs.
  • mice were treated with parathyroid hormone (PTH) (80 ⁇ g/kg/d) for 6 or 14 days.
  • a negative control group was treated with saline, a positive control group with granulocyte-colony stimulating factor (G-CSF) (200 ⁇ g/kg/d) for 5 days.
  • G-CSF granulocyte-colony stimulating factor
  • the results obtained in these experiments showed an increase of all subpopulations of bone marrow derived cells in the peripheral blood after stimulation with PTH.
  • the CD45 + /CD34 + subpopulations remained constant whereas the percentage of CD45 + /CD34 - subpopulations decreased after stimulation.
  • Serum levels of G-CSF but not SCF show increased values.
  • the present invention shows for the first time the role of parathyroid hormone as an effective stimulator for mobilization of bone marrow derived cells. Therefore, PTH is a promising substance for the repair of defect tissues, in particular ischemic tissues via mobilizing bone marrow derived cells as described herein.
  • the present inventors when addressing the question whether PTH beside its vasodilating effects regulate the bone marrow stem cell niche, so as to effect repair of tissue, in particular ischemic tissue as described herein, aimed to define survival, functional parameters as well as stem cell mobilization in a murine model of surgically induced myocardial infarction (MI) after treatment with PTH.
  • MI myocardial infarction
  • LAD left anterior descendens
  • pressure volume relationships were investigated in vivo using conductance catheters.
  • infarct size as well as cell proliferation was determined by BrdU and Ki67 incorporation.
  • stem cell mobilization was analyzed by FACS.
  • PTH treatment resulted in a significant improvement of survival post MI (60% vs. 40%).
  • FACS data on peripheral blood samples demonstrated stem cell mobilization 6 and 14 days after PTH treatment.
  • PTH treatment resulted in an improved myocardial function showing comparable values at day 6 (EF: 30% vs. 15%) and day 30 (EF: 29% vs. 15%).
  • Functional improvement was associated with a reduced peripheral resitance at day 6 (Arterial elastance Ea: 6,0 vs. 8,9 mmHg/ ⁇ l) and day 30 (Ea: 11 ,4 vs. 7,1 mmHg/ ⁇ l).
  • Infarct size was reduced at day 30 (23% vs.
  • PTH is not beneficial in the treatment of acute myocardial infarction which causes ischemia and, thus, organ defects.
  • the present invention discloses and demonstrates herein, in particular in the appended Examples, that PTH is preferably useful in the prevention and/or treatment of ischemia.
  • parathyroid hormone also abbreviated as “PTH” is sometimes also referred to as “parathormone” encompasses both naturally-occurring and synthetic forms of PTH and other forms of PTH, such as variants, analogs etc. as described herein below. It is to be understood that PTH is a polypeptide.
  • polypeptide when used herein means a peptide, a protein, or a polypeptide which are used interchangeable and which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds.
  • peptidomimetics of such proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine.
  • Peptides, oligopeptides and proteins may be termed polypeptides.
  • polypeptide and protein are often used interchangeably herein.
  • polypeptide also refers to, and does not exclude, modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
  • a modified PTH i.e. [Leu 27 ] cyclo (Glu 22 - Lys 26 )PTH (1-31) is a PTH form encompassed by the term PTH.
  • Naturally-occurring PTH is a secreted 84 amino acid product of the mammalian parathyroid gland that controls serum calcium levels through its action on various tissues, including bone.
  • PTH containing 84 amino acids is designated as PTH 1-84; see G.N. Hendy et al. (1981), Proc. Natl. Acad. Sci. USA 78, 7365 ; T. Kimura et al. (1983), Biochem. Biophys. Res. Commun. 114, 493 ; J. M. Zanelli et al. (1985), Endocrinology 117, 1962 or E. Wingender et al. (1989), J. Biol. Chem. 264, 4367 .
  • PTH is also disclosed in EP-B1 926 158 or EP-A1 1 059 933 .
  • PTH may be obtained by known recombinant or synthetic methods, such as described in U. S. Pat. Nos. 4, 086, 196 and 5, 556, 940 .
  • PTH can be obtained by peptide chemical synthesis which is ordinarily conducted.
  • the azide method, the acid chloride method, the azide anhydride method, the DCC method, the activated ester method, the carbon imidazole method or the oxidization-reduction method may be employed.
  • PTH when used herein includes a parathyroid hormone which is characterized by having the amino acid sequence shown in SEQ ID NO: 1.
  • SEQ ID NO: 1 shows the human PTH full-length peptide. The full-length peptide contains 115 amino acids. Amino acids 1-25 are believed to belong to a signal sequence, amino acids 26-115 are believed to belong to the PTH proprotein and amino acids 32 to 115 are believed to belong to the parathyroid hormone. Amino acids 32 to 115 of SEQ ID NO: 1 as described herein are regarded to constitute PTH (1-84) shown in SEQ ID NO: 2, wherein amino acid position 32 and 115 of SEQ ID NO: 1 correspond to positions 1 and 84 of PTH, respectively.
  • PTH (1-84) includes PTH(1-34) which corresponds to amino acid positions 32 to 65 of SEQ ID NO: 1.
  • PTH (1-34) shown in SEQ ID NO: 3 also amino acid position 32 of SEQ ID NO: 1 corresponds to amino acid position 1. The same holds true for the numbering of all PTHs described herein.
  • PTH from mouse (Accession No. NP 065648), rat (Accession No. NP 058740), chicken (Accession No. NP 990783), bovine (Accession No. NP 776379) or other mammals is contemplated to be employed by the pharmaceutical compositions for use in the medical treatment of the present application.
  • PTH "variants" wherein one or more amino acid residues are substituted, preferably conservatively substituted compared to said polypeptide and wherein said variant is preferably able to have PTH activity.
  • Any form of PTH that can be employed in the pharmaceutical compositions used in the method of treatment of the present invention has preferably PTH activity which is preferably characterized by the capability of PTH to bind to its receptor. Alternatively, PTH activity can be measured as is known in the art.
  • Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have no effect on the activity of the polypeptide.
  • Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, Science 247: (1990) 1306-1310 , wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicate that these positions are not critical for protein function.
  • positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. ( Cunningham and Wells, Science 244: (1989) 1081-1085 .) The resulting mutant molecules can then be tested for biological activity.
  • Tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and lie; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • amino acid substitutions may also increase protein or peptide stability.
  • amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence as well as substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D- amino acids or non-naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids.
  • PTH variants incorporate from 1 to 5 amino acid substitutions that improve PTH stability and half-life, such as the replacement of methionine residues at positions 8 and/or 18 with leucine or other hydrophobic amino acid that improves PTH stability against oxidation and the replacement of amino acids in the 25-27 region with trypsin-insensitive amino acids such as histidine or other amino acid that improves PTH stability against protease.
  • PTH fragments are characterized as having 84-n amino acids, wherein n is an integer from 1 to 56.
  • PTH fragments contain sequential deletions from the C-terminal e.g. fragments desirably contain at least the first 28 N-terminal residues, e.g. PTH (1-28), PTH (1-31), PTH (1-34), PTH (1-37), PTH (1-38) and PTH (1-41).
  • PTH (1-34) which is also known as teriparatide; see, for example S. H. Doppelt et al. (1981), Calcif. Tissue Int. 33, 649 ; R.Podbesek et al.
  • PTHrP parathyroid hormone-related peptide
  • a PTH peptide from any organism could be employed in the pharmaceutical compositions used in the method of treatment present application.
  • a PTH peptide from any organism can, for example, be identified by using sequence comparisons and/or alignments by employing means and methods known in the art, preferably those described herein and comparing and/or aligning (a) known PTH to/with a sequence suspected to be a PTH. For example, when a position in both of the two compared sequences is occupied by the same amino acid monomer subunit (for instance, if a position in each of two polypeptides is occupied by a lysine), then the respective molecules are identical at that position.
  • the percentage identity between two sequences is a function of the number of matching or identical positions shared by the two sequences divided by the number of positions compared x 100. For instance, if 6 of 10 of the positions in two sequences are matched or are identical, then the two sequences are 60% identical. By way of example, the amino acid sequences ALTSPY and AYTIWY share 50% homology (3 of the 6 total positions are matched). Generally, a comparison is made when two sequences are aligned to give maximum homology and/or identity. Such alignment can be provided using, for instance, the method of Needleman, J. Mol Biol. 48 (1970): 443-453 , implemented conveniently by computer programs such as the Align program (DNAstar, Inc.).
  • homologous sequences share identical or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence.
  • a "conservative substitution" of a residue in a reference sequence are those substitutions that are physically or functionally similar to the corresponding reference residues, e. g., that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like.
  • Particularly preferred conservative substitutions are those fulfilling the criteria defined for an " accepted point mutation" in Dayhoff et al., 5: Atlas of Protein Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed. Res. Foundation, Washington, D. C. (1978 ).
  • PTH does not only stand for a polypeptide having an amino acid sequence, but also stand for the nucleic acid molecules having nucleotide sequences encoding the amino acid sequence of PTH.
  • nucleic acid molecule when used herein encompasses any nucleic acid molecule having a nucleotide sequence of bases comprising purine- and pyrimidine bases which are comprised by said nucleic acid molecule, whereby said bases represent the primary structure of a nucleic acid molecule.
  • Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms, for example, PNA, and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • the polynucleotide of the present invention is preferably composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • the polynucleotide can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • “Modified” bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, the term “nucleic acid molecules" embraces chemically, enzymatically, or metabolically modified forms. Also nucleic acid sequences which hybridize to a nucleic acid sequence encoding a PTH can be used.
  • hybridizing sequences preferably refers to sequences which display a sequence identity of at least 65%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97, 98% or 99% identity with a nucleic acid sequence as described above.
  • Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001 ); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989 ), or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington DC, (1985 ).
  • the setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art.
  • the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as 0.1xSSC, 0.1% SDS at 65°C.
  • Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6xSSC, 1 % SDS at 65°C.
  • the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.
  • variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • Hybridizing nucleic acid molecules also comprise fragments of the above described molecules.
  • Such fragments may represent nucleic acid sequences as described herein.
  • nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules.
  • a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).
  • a solid support e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed.
  • complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • the sequence "A-G-T” binds to the complementary sequence "T-C-A”.
  • Complementarity between two single- stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules.
  • nucleic acid strands The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.
  • identity in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 65% identity, preferably, at least 70-95% identity, more preferably at least 95%, 96%, 97%, 98% or 99% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection.
  • Sequences having, for example, 65% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence.
  • Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program ( Thompson Nucl. Acids Res. 2 (1994), 4673-4680 ) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245 ), as known in the art.
  • the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity.
  • CLUSTALW CLUSTALW
  • the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10.
  • BLAST2.0 which stands for Basic Local Alignment Search Tool ( Altschul, Nucl. Acids Res. 25 (1997), 3389-3402 ; Altschul, J. Mol. Evol. 36 (1993), 290-300 ; Altschul, J. Mol. Biol. 215 (1990), 403-410 ), can be used to search for local sequence alignments.
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences.
  • the fundamental unit of BLAST algorithm output is the High- scoring Segment Pair (HSP).
  • An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
  • the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
  • the parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.
  • BLAST Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
  • the basis of the search is the product score which is defined as: % sequence identity ⁇ % maximum BLAST score 100 and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact.
  • nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above- described nucleic acid molecules may be used.
  • the term "being degenerate as a result of the genetic code” means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.
  • the complementary strand to the aforementioned and below mentioned nucleic acid molecules if they may be in a single-stranded form may be used.
  • the nucleic acid molecule may be any type of nucleic acid, e.g.
  • PNA peptide nucleic acid
  • a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254:1497 (1991 ); and Egholm et al., Nature 365:666 (1993 ), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases.
  • PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
  • the DNA may, for example, be genomic DNA or cDNA.
  • the RNA may be, e.g., mRNA.
  • the nucleic acid molecule may be natural, synthetic or semisynthetic or it may be a derivative, such as peptide nucleic acid ( Nielsen, Science 254 (1991), 1497-1500 ) or phosphorothioates.
  • the nucleic acid molecule may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination.
  • stem cells when used herein means that stem cells are mobilized from places within an animal or human body, e.g. from bone marrow so as to be present in the periphery, from where said stem cells can preferably migrate to other places within the animal or human body, preferably into tissue suffering from ischemia.
  • tissue suffering from ischemia includes cells, tissue and/or organs of the animal or human body which suffer from ischemia.
  • stem cells as used herein also refers to the ability to attract stem and/or progenitor cells from, e.g. the bone marrow preferably towards tissue suffering from ischemia.
  • stem cell homing is required to induce stem cell homing to injured tissue, e.g., myocardium.
  • Stem cells may bind SDF-1 via their CXCR4 receptor ( Petit (2002) Nat. Immunol. 687-694 ).
  • the aforementioned process is also understood as “homing”.
  • the term "homing” as used herein refers to the stem and/or progenitor cells' innate ability to travel preferably to the right place in the body.
  • said stem and/or progenitor cells travel to sites where organ defects/dysfunction caused by ischemia have taken/take place.
  • said stem cells are selected from the group consisting of CD34(+)/CD45(+) and CD34(-)/CD45(+) cells, each in combination with the subpopulations CD31(+), Sca-1 (+) or C-kit(+), multipotent adult progenitor cells (MAPC)1 endothelial progenitor cells (EPC) characterized by CD34(+), CD45(+), CD31(+), side population cells (SP) and lineage-negative stem cells, lin(-), c- kit(+).
  • the aforementioned stem cells may additionally express CXCR4.
  • the stem cells and/or progenitor cells are characterized by using FACS analysis as described in the appended Examples.
  • stem cells which express c-kit are efficiently mobilized (5-fold higher in comparison to a control).
  • the stem cells as described herein repair and/or regenerate tissue suffering from ischemia.
  • the terms "repairing” and “regenerating” as used herein relates to restore at least partially the former conditions of the defect organ tissue.
  • the indication "(+)” and “(-)” refer to results obtained by flow cytometry (FACS) analysis.
  • (+) or “positive” means that a defined protein, such as CD45, CD34 or CD31 , etc. is expressed on the surface of an analyzed cell, such as a stem cell or progenitor cell, etc.
  • (-) or “negative” means that a defined protein, such as CD34, CD31 etc. is not expressed on the surface of an analyzed cell, such as a stem cell or a progenitor cell, etc.
  • pharmaceutical composition relates to a composition comprising PTH or specific fragments thereof. In some embodiments described herein, said pharmaceutical composition also comprises G-CSF or a G-CSF fragment thereof.
  • said pharmaceutical composition also comprises a DPP IV antagonist/inhibitor. It has also been observed that physical exercise is beneficial for mobilizing stem cells. Accordingly, it is envisaged that the medical uses may be accompanied by physical exercise and any other measure which is suitable to mobilize stem cells.
  • a pharmaceutical composition comprises a therapeutically effective amount of PTH or specific fragments thereof and, optionally, a pharmaceutically acceptable carrier.
  • Said pharmaceutical composition may also comprise one or more of the following compounds, e.g., GM-CSF, SCF, IL-3, IL-6, EPO, VEGF, a DPP IV antagonist, statins or thiolactones. As detailed herein, these factors may also be employed in combination in the herein disclosed (medical or pharmaceutical) uses and methods of this invention.
  • the pharmaceutical composition may be administered with a physiologically acceptable carrier to a patient, as described herein.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin. Such compositions will contain a therapeutically effective amount of the aforementioned compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
  • the pharmaceutical composition is suitable for administration to a patient.
  • patient means an individual in need of a treatment, preferably in need of a treatment of a tissue suffering from ischemia.
  • the patient is a vertebrate, even more preferred a mammal, particularly preferred a human.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to mammals, preferably vertebrates and more preferably human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the pharmaceutical composition can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • In vitro assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder should be decided according to the judgement of the practitioner and each patient's circumstances. Moreover, for example, the following factors concerning the precise dose may also be taken into account: patient's size, body surface area, age, sex, general health, and other drugs being administered concurrently. Therefore, it is well known in the art that the dosage regimen will be determined by the attending physician and clinical factors.
  • Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the dosage regimen of the pharmaceutical composition is to be administered to the patient ranges preferably from 0.1 to 200, 0.1 to 150 or 0.1 to 100 ⁇ g per kg body weight, more preferably from 1 to 100 ⁇ g per kg body weight, even more preferably from 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 ⁇ g per kg body weight d. s. c. over a period of at least 1 day, preferably at least 2 days, more preferably at least 3 days, even more preferably at least 4 days, particularly preferred at least 5 days, even more particularly preferred at least 6 days, 10 days, 14 days, 21 days, 28 days or 30 days.
  • any other suitable dosage regimen is envisaged which may be determined as described herein.
  • the administration of the candidate agents can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, intrabronchially, transdermally, intranodally, intradermally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, intraocularly, vaginally, rectally or topically.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the pharmaceutical composition is administered subcutaneously.
  • the pharmaceutical composition is administered via routes of administration as described supra and infra.
  • compositions of the present invention also pertain to pharmaceutical compositions comprising, apart from PTH or specific fragments thereof, G-CSF fragments and a DPP IV antagonist.
  • This embodiment applies, mutatis mutandis, for the herein disclosed (pharmaceutical and/or medical) uses and methods. Accordingly, also the combinatory/combinational uses of these factors and antagonists are envisaged.
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • G-CSF polypeptide having an additional methionine residue at its N-terminus is known.
  • the function of G-CSF can be tested by methods known in the art, for example, described in PCT/EP2004/01 2036 ( WO/2005/049062 ).
  • Said term also encompasses G-CSF polypeptides from preparations well known in the art, either from natural sources or preferably produced by recombinant means. Multiple forms of natural or recombinant human, mouse or rat G-CSF are known in the art. It is envisaged that the G-CSF or fragment thereof used in the context of the present invention is of pharmaceutical grade suitable for administration to patients as described infra.
  • G-CSF polypeptides comprising an amino acid sequence at least 70%, 80%, 90%, 95%, 97% or 99% identical to the G-CSF polypeptide which is known in the art and has preferably G-CSF activity as described in WO/2005/049062 .
  • G-CSF fragment when used in the context of the present invention means fragments of G-CSF polypeptides having G-CSF activity.
  • the amino acid sequence of G-CSF and of corresponding variants is known in the art and published in Nagata (1986), Nature 319:415-418 .
  • said G-GSF fragments comprise portions of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 or 170 amino acid residues of the G-CSF protein.
  • said fragments are biologically active fragments, i.e. fragments which have G-CSF activity.
  • said fragments are capable to, e.g., mobilize multipotent stem cells, improve cardiac function or reduce mortality after acute myocardial.
  • Particularly preferred G-CSF activity can be determined by its ability to induce colony formation, as reported in the literature by Bodine (1993), Blood 82:445-55 ; Bodine (1994), Blood 84, 1482-91 .
  • G-CSF variants are envisaged. Accordingly, G-CSF variants are such variants as described in the context of PTH variants.
  • treatment and “treating” are used herein to generally mean obtaining a desired pharmaceutical and/or physiological effect.
  • the effect is therapeutic in terms of partially or completely curing ischemia.
  • treatment covers any treatment of tissue suffering from ischemia and/or organ defects and/or dysfunction caused by ischemia in a mammal, particularly a vertebrate and more preferably a human, and includes regenerating and/or repairing suffering from ischemia and/or organ or tissue dysfunction.
  • the pharmaceutical composition of the present invention is suitable for the treatment of ischemia.
  • prevention means to obtain a protective effect on a tissue which is already suffering from ischemia so as to prevent further damage and/or a protective effect on a tissue which is at a risk of suffering from ischemia.
  • the pharmaceutical composition of the present invention for the purpose of preventing ischemia may preferably be administered to a subject who is at a risk of ischemia of the heart.
  • ischemia as used herein relates to a condition that may occur in any tissue and/or organ that is suffering a lack of oxygen supply and/or supply with metabolites which occurs when there is an imbalance between oxygen supply and demand, due to inadequate perfusion, e.g., caused by atherosclerosis, restenotic lesions, anemia, stroke or clogged arteries just to name a few , that leads to insufficient oxygen to tissues such as, for example, the heart or brain.
  • medical interventions such as the interruption of the blood flow, e.g., during bypass surgery may lead to ischemia.
  • Said term also encompasses the two most common types of ischemia; i.e.
  • Cardiac ischemia includes a broad variety of conditions, from silent ischemia to stable or unstable angina to myocardial infarction (AMI or "heart attack"). Cerebral ischemia includes prolonged cerebral ischemic syndromes to completed stroke or cerebral infarction.
  • ischemia is not limited to the aforementioned organs or tissues, respectively, since it may occur in any organ that is suffering a lack of oxygen supply and/or supply with metabolites. In the context of the present invention, ischemia causes organ dysfunction and/or organ defects.
  • surgical or interventional procedure relates to a surgical and/or interventional procedure which is suitable to improve organ function, to improve blood flow in defect organ tissue and/or to induce revascularization as thrombolysis (either systemic or local via catheter delivery), balloon angioplasty, stenting, coronary, carotid or peripheral bypass surgery, endatherectomy or ventriculo-coronary stenting.
  • administered means administration of a therapeutically effective dose of a pharmaceutical composition.
  • said therapeutically effective dose is administered to a patient who has tissue suffering from ischemia.
  • Particularly preferred said therapeutically effective dose is administered to a patient suffering from organ defects and/or dysfunction caused by ischemia.
  • therapeutically effective amount is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.
  • compositions are applicable to both human therapy and veterinary applications.
  • the compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein.
  • the compounds may be formulated in a variety of ways as discussed below.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %.
  • the agents maybe administered alone or in combination with other treatments.
  • the administration of the pharmaceutical composition can be done in a variety of ways as discussed above.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g; and as described supra however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the dosages are preferably given once, twice, trice, four times or five times a day for the period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 or 30 days, however, during progression of the treatment the dosages can be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., daily divided into multiple applications. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively.
  • the pharmaceutical compositions are employed in co-therapy approaches, i.e. in coadministration with other medicaments or drugs, for example other drugs for preventing, treating and/or ameliorating ischemia. It is also preferred that the pharmaceutical composition is co-administered with GM-CSF (granuloyte macrophage colony stimulating factor), SCF (stem cell factor), IL-3 (interleukin-3) and/or IL-6 (interleukin-6), EPO, VEGF or a fragment thereof, statins, thiolactones and/or with a DPP IV antagonist.
  • GM-CSF granuloyte macrophage colony stimulating factor
  • SCF stem cell factor
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • EPO VEGF or a fragment thereof, statins, thiolactones and/or with a DPP IV antagonist.
  • the pharmaceutical composition of the present invention is co-administered with G-CSF or a G-CSF fragment and/or with a DPP IV antagonist.
  • a DPP IV antagonist may be administered in combination with one or more of the aforementioned substances.
  • the pharmaceutical composition can be administered before a surgical or interventional procedure.
  • the term "before said surgical or interventional measure" when used in the context of the present invention means that the pharmaceutical composition of the present invention is to be administered before one day, preferably two days, more preferably three days and even more preferably four or five days of said surgical or interventional measure, for example, to prevent ischemic disease as described herein.
  • the pharmaceutical composition of the present invention can also be administered during a surgical or interventional procedure.
  • the term "during said surgical or interventional measure" when used in the context of the present invention means that the pharmaceutical composition can to be administered during a surgical or interventional procedure. Additionally, the pharmaceutical composition can be administered after a surgical or interventional procedure.
  • the pharmaceutical composition when used in the context of the present invention means that the pharmaceutical composition can be administered preferably immediately after said surgical or interventional measure. Particularly preferred, it can be be administered preferably immediately after said surgical or interventional measure or, alternatively, after at least 1 hour, preferably after at least 6, 12, 24 or 48 hours.
  • the pharmaceutical composition of the present invention after it has been administered before, during or after a surgical or interventional measure it can be further administered for the period of time as described above so as to preferably prevent and/or treat ischemic heart diseases.
  • the ischemic heart diseases treated with the pharmaceutical composition is selected from myocardial ischemia, silent ischemia and stable or unstable angina.
  • the myocardial ischemia which is treated with the pharmaceutical composition are caused by heart failure, hypertension, coronary artery disease (CAD), myocardial infarction, thrombo-embolic events, trauma and/or surgical procedures.
  • Other causes of ischemia which can be treated with the pharmaceutical composition of the invention are:
  • Liver ischemia or retinal ischemia which is treated with the pharmaceutical composition is caused by thrombo-embolic events, malformation of blood-supplying vessels, trauma and/or surgical procedures.
  • Peripheral muscle tissue ischemia or spinal cord ischemia which is treated with the pharmaceutical composition is caused by thromboembolic events, atherosclerosis, malformation of blood-supplying vessels, trauma and/or surgical procedures.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising PTH or specific fragments thereof and/or G-CSF or a G-CSF fragment for use in the treatment of ischemic heart disease.
  • the present invention relates to a pharmaceutical composition comprising PTH or specific fragments thereof and/or a DPP IV antagonist for use in the treatment of ischemic heart diesease.
  • a pharmaceutical composition comprising PTH or specific fragments thereof and/or a DPP IV antagonist and/or any of the compounds mentioned herein, e.g., GM-CSF, SCF, IL-3, IL-6, statins, thiolactones and/or EPO can be used.
  • the findings observed in the murine model with respect to stem cell mobilization are summarized: in our study we examined the potency of PTH to mobilize bone marrow derived cells and investigated the changes in the composition of cells in bone marrow after administration of PTH. Furthermore, we analysed the serum levels of cytokines as possible effectors.
  • N-cadherin-mediated adhesion may link to the Wnt-LEF-1-Notch1 pathway through ⁇ -catenin signaling 21, 22 .
  • Our results indicate that these mechanism may only effect on CD45 + /CD34 + cells resulting in a constant level of these cells in bone marrow.
  • cytokines like G-CSF, SCF, VEGF or SDF-1 are known to stimulate bone marrow causing a release of stem cells into the circulation 2, 4, 7 .
  • PTH serum levels of G-CSF and SCF, factors employed in several studies aiming to mobilize stem cells 2, 23-26 .
  • After administration of PTH we found a strong increased concentration of G-CSF, whereas a slight but not significant decrease of SCF was detectable.
  • Osteoblasts that are among other structural cells in the hematopoietic stem cell niche, can be activated by PTH and are able to produces growth factors 27-29 .
  • Our data suggest an indirect mobilizing effect of PTH by stimulating osteoblasts to produce cytokines such as G-CSF.
  • SCF is negative regulated by PTH, however the pathway and function is not yet known and remains to be elucidated.
  • G-CSF treatment did not show a stabilizing effect on CD45 + /CD34 + cells, but resulted in a significant decrease of these cell in bone marrow as a sign of cell release without self-renewal.
  • G-CSF and PTH treatment caused a decrease of CD45 + /CD34 - cells in bone marrow at comparable levels, but with different intensities on different subtypes.
  • the results suggest an independent effect of PTH on bone marrow derived cells.
  • Our study shows for the first time the ability of PTH to mobilize bone marrow derived cells. The mechanism for cell mobilization might occur in part through the endogenous release of cytokines such as G-CSF but not SCF.
  • PTH shows a stabilizing effect in bone marrow. This effect of PTH seems to act especially on CD34 + cells.
  • the main findings of the present invention with respect to the murine model for ischemia are: 1) a beneficial effect on myocardial function and survival which was related to 2) an increased mobilization of stem cells into peripheral blood 3) a reduced arterial load and a 4) a beneficial effect on postinfarct remodelling characterized by a higher rate of endothelial proliferation and a reduced decline of the LV anterior wall.
  • PTH treatment induces stem cell mobilization into the peripheral blood
  • PTH(1-34) treatment was shown to increase myocardial blood flow by reducing coronary artery resistance in pigs in normal and stunned myocardium after intracoronary infusion of PTH(1-34) in a cumulative dose between 12 and 18 ⁇ g 32 .
  • the effect of an intermittent application of PTH after MI was not studied yet.
  • the application of 80 ⁇ g/kg/d of PTH resulted in a significant reduction of arterial resistance at day 6 and at day 30 suggesting early vasorelaxing effects in the arterial system including the coronary vasculature.
  • the highest mortality appeared in the early 6 days after MI this data might be related to others showing a prevention from cardiogenic shocks after PTH treatment 47 .
  • PTH (1-34) treatment was shown to increase myocardial blood flow by reducing coronary artery resistance in pigs and rats in normal and stunned myocardium after intracoronary infusion of PTH (1-34) in a cumulative dose between 12 and 18 ⁇ g.
  • Another experimental study which subjected male Sprague-Dawley rats to permanent middle cerebral artery occlusion showed, that PTHrP(1-34) peptide treatment significantly decreased cortical infarct size up to 47%.
  • PTH(1-34) The impact of PTH(1-34) on angiogenesis and cell survival is mediated via the VEGF and IGF-1 axis.
  • IGF-1R insulin receptor substrates
  • IRS-1 and IRS-2 phosphorylation of the insulin receptor substrates
  • Tyrosine-phosphorylated IRSs interact with cytoplasmic proteins with src homology 2 (SH2) domains, such as phosphatidylinositol 3-kinase (PI3K).
  • PI3K activation then leads to the transduction of the functional effects of IGF-1, such as enhanced glucose transport, enhanced cardiomyocyte contractility, and the inhibition of programmed cell death (apoptosis).
  • PI3K phosphatidylinositol 3-kinase
  • CD34 + bone marrow cells The mobilization and transfusion of CD34 + bone marrow cells contributes to enhanced neovascularization.
  • Isolated CD34 + blood and bone marrow cells express and secret angiogenic growth factors including VEGF, HGF and IGF-1.
  • VEGF vascular endothelial growth factor
  • HGF vascular endothelial growth factor
  • IGF-1 angiogenic growth factors
  • VEGF-A und VEGF-R1 is a direct effect of PTH or results from mobilized infiltrated blood cells secreting VEGF protein.
  • Immunostaining of hearts 2 and 6 days after MI revealed a high number of leucocytes and monocytes in the granulation tissue which stained positive for VEGF protein supporting the thesis that angiogenic factors have been carried to the site of ischemia.
  • cardioprotective c-kit+ cells are derived from the bone marrow and act on ischemic myocardium by enhanced neovascularization through angiogenic cytokines like VEGF-A.
  • VEGFR1 is a functional receptor on monocyte/macrophage and neutrophils which could be recruited to sides of ischemia by VEGF-A.
  • the VEGFR1-expressing monocyte/macrophages were not able to incorporate into vascular structures but could release angiogenic factors like VEGF-A and induce neovascularization.
  • Another source for the enriched growth factor expression could be the enhanced migration of CD45 + /Sca-1 + cells after PTH application.
  • IGF-1 Another known factor which is secreted from infiltrated blood cells is IGF-1.
  • IGF-1 receptor protein was upregulated basically on cardiomyocytes at the borderzone at day 2 and at day 6 which was associated with an increased amount of neovascularization and a reduced number of apoptotic cardiomyocytes.
  • IGF-1R cell surface receptor
  • IRS-1 and IRS-2 Tyrosine-phosphorylated IRSs interact with phosphatidylinositol 3-kinase (PI3K).
  • IGF-1 interleukin-1-1
  • PI3K activation leads to the transduction of the functional effects of IGF-1, such as enhanced neovascularization, enhanced cardiomyocyte contractility, and the inhibition of programmed cell death (apoptosis).
  • IGF-1 deficient mice showed an impaired cardiac remodeling after myocardial infarction whereas overexpression of IGF-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy.
  • IGF-1 increased VEGF mRNA and protein expression by 2 fold and recently it was shown that IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and -independent pathways.
  • IGF-1 stimulated VEGF promoter activity was PI3-K/Akt/mTOR dependent.
  • IGF-1 receptor was highly expressed basically on cardiomyocytes at the infarct borderzone. As IGF-1 mRNA was also upregulated this could mean a higher sensitivity of borderzone cardiomyocytes to cardioprotective IGF-1 effects like reduced apoptosis and increased neovascularization.
  • CD45 + /CD34neg cells only expressed 10% CXCR4 on their surface indicating a CXCR4-SDF-1 independent mechanism of cell migration to the heart.
  • the relevance for the increased homing of the CD34 + /CXCR4 + cell subset is supported by our finding that the homing factor SDF-1 ⁇ is upregulated in the ischemic myocardium 48 hours after PTH treatment.
  • the homing of CD45/CD34/CXCR4 positive cells was also seen in saline treated and sham operated animals.
  • the migration was less pronounced especially in sham operated mice and was related to a reduced SDF-1 expression 48 hours after MI (compared with PTH treatment) showing a positive relation between SDF-1 expression and the migration of CD45/CD34/CXCR4 positive stem cells to the heart.
  • SDF-1 ⁇ overexpression in a chronic model of cardiac ischemia led to an increased infiltration of CD34 and c-kit positive stem cells to the heart but the CXCR4 fraction was not analyzed.
  • An 80% increased homing of intravenous infused genetically marked Lin - bone marrow cells to the heart 48 hours after MI was shown.
  • Administration of AMD3100 which specifically blocks binding of SDF-1 to its endogenous receptor CXCR4, diminished the bone marrow derived cell recruitment after MI by 64%.
  • PTH treatment after myocardial infarction resulted in the mobilization and migration of stem cells and preservation of cardiac function by increased angiogenesis and cell survival via VEGF-A and IGF-1 mediated mechanisms which could be explained by an enhanced migration of angiogenic CD45/CD34/CXCR4 positive stem cells.
  • PTH 1-34
  • parathyroid hormone might be an interesting supplement or alternative to other stem cell modulating agents like G-CSF or GM-CSF in ischemic heart disease.
  • results and considerations could be applicable as regards PTHrP or a combination of PTH and PTHrP. Accordingly, these results and considerations may be generalizable insofar as PTH and/or PTHrP is/are useful for recruiting of stem cells from the bone marrow into the periphery and, further, is/are useful for the prevention and/or treatment of ischemia.
  • FITC fluorescein-isothiocyanate
  • PE phycoerythrin
  • PerCP peridinin-chlorphyll-protein conjugated monoclonal antibodies: CD45-PerCP, C34-FITC, CD31-PE, Sca-1-PE and c-kit-PE (all from BD Pharmingen). Isotype-identical antibodies (BD Pharmingen) served as controls.
  • Cells were analyzed by 3-color flow cytometry using a Coulter® Epics® XL-MCLTM flow cytometer (Beckman Coulter). Each analysis included 20000 events.
  • the absolute number of antigen-positive cells per ml of whole blood was calculated by multiplying the percentage of antigen-positive cells with the total number of mononuclear cells per ml of blood detected by the conventional hematological cell analyzer.
  • the number of lymphocytes together with the number of monocytes comprised the total number of mononuclear cells.
  • G-CSF and SCF serum levels were determined by ELISA (Mouse G-CSF and Mouse SCF, RayBiotech).
  • CD45 + /CD34 + cells CD45 + /CD34 + /CD31 + , CD45 + /CD34 + /Sca-1 + , CD45 + /CD34 + /c-kit +
  • CD45 + /CD34 - cells CD45 + /CD34 - /CD31 + , CD45 + /CD34 - /Sca-1 + , CD45 + /CD34 - /c-kit +
  • Example 7 (CD45 + /CD34 + and CD45 + /CD34 - cell populations in peripheral blood after G-CSF treatment)
  • Example 8 (CD45 + /CD34 + and CD45 + /CD34 - cell populations in bone marrow after PTH treatment)
  • Example 9 (CD45 + /CD34 + and CD45 + /CD34- cell populations in bone marrow after G-CSF treatment)
  • G-CSF treatment resulted in a significant decrease of all subtypes of CD45 + CD34 + cells compared to the control group ( Fig 2A ).
  • CD45 + /CD34 - cells are significantly increased in bone marrow after G-CSF treatment (18.9% increase compared to saline treated mice, p ⁇ 0.001), whereas all investigated subtypes of CD45 + /CD34 - cells are significantly decreased ( Fig 2 B) .
  • Serum levels of G-CSF analysed by ELISA were significantly elevated at day 6 of PTH stimulation (2171.0 ⁇ 93.6 pg/ml vs. 6035.5 ⁇ 1318.4 pg/ml, p ⁇ 0.05).
  • SCF showed significant decreased levels at day 6 of PTH stimulation (670.4 ⁇ 19.1 pg/ml vs. 594.6 ⁇ 26.8 pg/ml, p ⁇ 0.05).
  • Myocardial infarction was induced in male C57BL/6 mice 8-12 weeks of age by surgical occlusion of the left anterior descending artery (LAD) through a left anterolateral approach.
  • Mice were anesthetized by intraperitoneal injection of a mixture of 100 mg/kg ketamine (Sigma Chemical Co., St. Louis, MO) and 5 mg/kg Xylazine (Sigma), intubated and artificially ventilated by a mouse ventilator (HUGO SACHS, March, Germany) with 200 strokes/min and 200 ⁇ l/stroke.
  • ketamine Sigma Chemical Co., St. Louis, MO
  • Xylazine Sigma
  • Animal care and all experimental procedures were performed in strict accordance to the German and National Institutes of Health animal legislation guidelines and were approved by the local animal care and use committees.
  • PBS phosphate-buffered saline
  • FITC fluorescein-isothiocyanate
  • PE phycoerythrin
  • PerCP peridinin-chlorophyll-protein conjugated monoclonal antibodies: CD45-PerCP, CD34-FITC, CD31-PE, Sca-1-PE and c-kit-PE (all from BD Pharmingen).
  • Matching isotype antibodies (BD Pharmingen) served as controls.
  • Cells were analyzed by 3-color flow cytometry using a Coulter ® Epics ® XL-MCL TM flow cytometer (Beckman Coulter). Each analysis included 20000 events.
  • Infarct size was determined as area of infarction (Al) correlated to the area of the left ventricle (including LV-septum) in four different slices from the base to the apex of a heart.
  • Total infarct size was calculated by multiplication of the mean percentage value of the circular infarct area with the quotient: vertical extension of the infarct area / total ventricular extension. Wall thickness was measured by taking the average length of five segments along radii from the centre of the left ventricle through the thinnest points of the free LV wall and the septal wall. All studies were performed by a blinded pathologist.
  • Pre-treatment was performed for 30 minutes (microwave 750 W) using TRS 6 (Dako) for CD45, Glykol (biologo) for CD34, Retrievagen A (BD Pharmingen) for BrdU, and citrate buffer (10 mM, pH 6.0) for Ki67.
  • Doublestaining was performed for CD31 and Ki67 using an Avidin-Biotinylated enzyme Comlex-Goat IgG detection system and diaminobenzidine as chromogen and the APAAP-Rat system and chromogen red (all from Dako), respectively.
  • Quantitative assessments a) granulation tissue: the number of BrdU and Ki76 positive nuclei were related to the total number of nuclei quantified in the granulation tissue. b) arterioles: only arterioles with high proliferative activity were enclosed.
  • Results were expressed as mean ⁇ S.E.M.
  • mice 4 weeks after myocardial infarction, rPTH (1-34) treated mice showed a significant increase in the survival rate compared to saline treated animals (60.0% vs. 40.2%). Mortality amongst untreated animals was very high within the first six days after myocardial infarction, whereas mice surviving the first 6 days after MI showed a lower mortality in both groups ( Fig.4 ).
  • Ejection fraction (EF: 29.0 ⁇ 7.1% vs. 14.5 ⁇ 0.9%, p ⁇ 0.001, Fig. 7 ), cardiac output (3810 ⁇ 205 vs. 2690 ⁇ 329, Fig. 8 ) and stroke work (382 ⁇ 59 vs. 258 ⁇ 27 mmHg x ⁇ l, p ⁇ 0.05, Fig. 9 ) were significantly improved.
  • cardiac output 3810 ⁇ 205 vs. 2690 ⁇ 329, Fig. 8
  • stroke work (382 ⁇ 59 vs. 258 ⁇ 27 mmHg x ⁇ l, p ⁇ 0.05, Fig. 9 ) were significantly improved.
  • diastolic relaxation tends to be improved after PTH treatment reflected by an accelerated diastolic relaxation (Tau Glantz: 12.0 ⁇ 0.8 vs.14.81 ⁇ 1.5 ms).
  • enddiastolic volumes (EDV: 34.4 ⁇ 6.1 vs. 41.3 ⁇ 3.2) were no significantly reduced in PTH(1-34) treated animals (Table 2).
  • infarct composition was altered with a significantly lower cellular density (4383 ⁇ 409 vs.
  • Ki67 positive arterioles revealed an increased expression of ICAM-1, which was associated with a pronounced infiltration of CD45 positive cells.
  • CD34 staining was observed on endothelial cells of capillaries and veins and the adventitia of arteries and further stromal cells, but not on endothelial cells of arterioles or infiltrating cells. Furthermore, there was no obvious difference either in strength of staining or in cell types staining positive for CD34 between G-CSF treated and saline treated mice (data not shown).
  • Example 22 (Improvement of cardiac function after myocardial infarction by increasing homing-capacity via DPP-IV-inhibition)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Claims (15)

  1. Pharmazeutische Zusammensetzung, die Parathormon (PTH) oder PTH-Fragmente mit 84-n Aminosäuren umfasst, wobei n für eine ganze Zahl von 1 bis 56 steht und wobei die Fragmente die ersten 28 n-terminalen Aminosäurereste enthalten, zur Verwendung bei der Behandlung von ischämischen Herzerkrankungen.
  2. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der vorstehenden Definition umfasst, zur Verwendung nach Anspruch 1, wobei das Fragment ausgewählt ist aus der Gruppe bestehend aus PTH(1-28), PTH(1-31), PTH(1-34), PTH(1-37), PTH(1-38) und PTH(1-41).
  3. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach Anspruch 1 oder 2, wobei die ischämische Herzerkrankung ausgewählt ist aus der Gruppe bestehend aus stiller Ischämie, stabiler oder instabiler Angina und Myokardischämie.
  4. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, zur Verwendung bei der Behandlung eines Myokardinfarkts.
  5. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche zur Rekrutierung von Stammzellen in ischämisches und/oder apoptotisches Gewebe, wobei die Stammzellen ausgewählt sind aus der Gruppe bestehend aus CD34(+)/CD45(+)- und CD34(-)/CD45(+)-Zellen, jeweils in Kombination mit den Subpopulationen CD31(+), Sca-1(+) oder C-kit(+), multipotenten adulten Progenitorzellen (MAPC), durch CD34(+), CD45(+), CD31(+) charakterisierten endothelialen Progenitorzellen (EPC), SP (Seitenpopulation)-Zellen sowie Lineage-negativen Stammzellen, lin(-), c-kit(+).
  6. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, wobei die pharmazeutische Zusammensetzung über einen Zeitraum von mindestens einem Tag mindestens ein Mal pro Tag verabreicht wird.
  7. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, wobei die pharmazeutische Zusammensetzung subkutan verabreicht wird.
  8. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, wobei es sich bei der ischämischen Herzerkrankung um eine durch Herzversagen hervorgerufene Myokardischämie, Hypertonie, koronare Herzerkrankung (CAD), Myokardinfarkt, thromboembolische Ereignisse, Trauma, chirurgische und/oder interventionelle Maßnahmen handelt.
  9. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach Anspruch 8, wobei es sich bei der chirurgischen oder interventionellen Maßnahme um eine Maßnahme zur Wiederherstellung des Blutflusses handelt, die ausgewählt ist aus der Gruppe bestehend aus Thrombolyse, Ballonangioplastie, Stenting, einer koronaren oder peripheren Bypass-Operation und ventrikulokoronarem Stenting.
  10. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, wobei die pharmazeutische Zusammensetzung zur Verabreichung in Kombination mit G-CSF oder einem G-CSF-Fragment vorgesehen ist.
  11. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, wobei es sich bei dem PTH-Fragment um PTH(1-34) handelt.
  12. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, wobei die pharmazeutische Zusammensetzung des Weiteren einen DPP IV-Antagonisten umfasst.
  13. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2 umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche, wobei es sich bei der ischämischen Herzerkrankung um eine durch Myokardinfarkt hervorgerufene Myokardischämie handelt.
  14. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente gemäß der Definition in Anspruch 1 oder 2, G-CSF oder G-CSF-Fragmente sowie einen DPP IV-Antagonisten umfasst, zur Verwendung nach einem der vorhergehenden Ansprüche.
  15. Pharmazeutische Zusammensetzung, die PTH oder PTH-Fragmente umfasst, nach Anspruch 12 oder 13 oder pharmazeutische Zusammensetzung nach Anspruch 14, wobei der DPP IV-Antagonist ausgewählt ist aus der Gruppe bestehend aus Diprotin A, Vildagliptin und Sitagliptin, zur Verwendung nach einem der vorhergehenden Ansprüche.
EP07724213.9A 2006-04-19 2007-04-12 Parathormon (pth) zur verwendung in der behandlung der ischämie Active EP2007416B1 (de)

Priority Applications (2)

Application Number Priority Date Filing Date Title
SI200731997T SI2007416T1 (en) 2006-04-19 2007-04-12 Parathyroid hormone (PTH) for use in the treatment of ischemia
PL07724213T PL2007416T3 (pl) 2006-04-19 2007-04-12 Parathormon (PTH) do zastosowania w leczeniu niedokrwienia

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US79294306P 2006-04-19 2006-04-19
PCT/EP2007/003272 WO2007118680A2 (en) 2006-04-19 2007-04-12 Remedies for ischemia

Publications (2)

Publication Number Publication Date
EP2007416A2 EP2007416A2 (de) 2008-12-31
EP2007416B1 true EP2007416B1 (de) 2017-09-27

Family

ID=38609853

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07724213.9A Active EP2007416B1 (de) 2006-04-19 2007-04-12 Parathormon (pth) zur verwendung in der behandlung der ischämie

Country Status (11)

Country Link
US (1) US8207116B2 (de)
EP (1) EP2007416B1 (de)
CY (1) CY1119819T1 (de)
DK (1) DK2007416T3 (de)
ES (1) ES2654251T3 (de)
HU (1) HUE035446T2 (de)
LT (1) LT2007416T (de)
PL (1) PL2007416T3 (de)
PT (1) PT2007416T (de)
SI (1) SI2007416T1 (de)
WO (1) WO2007118680A2 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2138572A1 (de) * 2008-06-26 2009-12-30 t2cure GmbH Mesoangioblastenartige Zellen sowie Verfahren und Anwendungen in Zusammenhang damit
WO2010045229A2 (en) 2008-10-13 2010-04-22 University Of Rochester Protecting and repairing cartilage and musculoskeletal soft tissues
GB201619861D0 (en) * 2016-11-24 2017-01-11 Narodden Salomon Treatments for heart failure and cardiac ischaemic reperfusion injury
JP7300719B2 (ja) * 2017-04-04 2023-06-30 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー 成体多能性幹細胞の調製、拡大および使用
US11845956B2 (en) 2018-09-21 2023-12-19 APstem Therapeutics, Inc. Human pluripotent adult stem cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005049022A2 (en) * 2003-11-17 2005-06-02 Novartis Ag Use of dipeptidyl peptidase iv inhibitors

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001278714A1 (en) * 2000-08-11 2002-02-25 Chugai Seiyaku Kabushiki Kaisha Vascular relaxation agents
US20040161412A1 (en) * 2002-08-22 2004-08-19 The Cleveland Clinic Foundation Cell-based VEGF delivery
EP1649008A2 (de) * 2003-07-16 2006-04-26 Boston Scientific Limited Ausgerichtete gerüste zur verbesserten myokardregeneration
EP1527785A1 (de) 2003-10-27 2005-05-04 Ludwig-Maximilians-Universität München Verwendung von G-CSF zur Behandlung von Ischemien
US20070021336A1 (en) * 2004-12-24 2007-01-25 Christen Anderson Use of GLP-1 and agonists thereof to prevent cardiac myocyte apoptosis
EP1841448A2 (de) * 2004-12-30 2007-10-10 Diakine Therapeutics, Inc. Pharmazeutische zusammensetzungen und vefahren zur wiederherstellung der betazellmasse und funktion

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005049022A2 (en) * 2003-11-17 2005-06-02 Novartis Ag Use of dipeptidyl peptidase iv inhibitors

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"TERIPARATIDE (PTH(1-34))", FDA, DRUGS DATABASE, 14 February 2013 (2013-02-14), pages 1 - 9 *
J. T POTTS: "Parathyroid hormone: past and present", JOURNAL OF ENDOCRINOLOGY, vol. 187, no. 3, 1 December 2005 (2005-12-01), GB, pages 311 - 325, XP055358962, ISSN: 0022-0795, DOI: 10.1677/joe.1.06057 *
J. T. POTTS ET AL: "Synthesis of a Biologically Active N-Terminal Tetratriacontapeptide of Parathyroid Hormone", PROCEEDINGS NATIONAL ACADEMY OF SCIENCES PNAS, vol. 68, no. 1, 1 January 1971 (1971-01-01), US, pages 63 - 67, XP055358961, ISSN: 0027-8424, DOI: 10.1073/pnas.68.1.63 *
M.-M. ZARUBA ET AL: "Parathyroid hormone treatment after myocardial infarction promotes cardiac repair by enhanced neovascularization and cell survival", CARDIOVASCULAR RESEARCH, vol. 77, no. 4, 12 December 2007 (2007-12-12), GB, pages 722 - 731, XP055265987, ISSN: 0008-6363, DOI: 10.1093/cvr/cvm080 *
SHIMIZU M ET AL: "MINIMIZATION OF PARATHYROID HORMONE NOVEL AMINO-TERMINAL PARATHYROID HORMONE FRAGMENTS WITH ENHANCED POTENCY IN ACTIVATING THE TYPE-1 PARATHYROID HORMONE RECEPTOR", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 275, no. 29, 21 July 2000 (2000-07-21), pages 21836 - 21843, XP002939322, ISSN: 0021-9258, DOI: 10.1074/JBC.M909861199 *

Also Published As

Publication number Publication date
PL2007416T3 (pl) 2018-05-30
DK2007416T3 (en) 2017-12-11
US8207116B2 (en) 2012-06-26
US20090286730A1 (en) 2009-11-19
PT2007416T (pt) 2018-01-03
WO2007118680A3 (en) 2008-02-14
CY1119819T1 (el) 2018-06-27
SI2007416T1 (en) 2018-02-28
LT2007416T (lt) 2018-02-12
ES2654251T3 (es) 2018-02-12
EP2007416A2 (de) 2008-12-31
WO2007118680A2 (en) 2007-10-25
HUE035446T2 (en) 2018-05-02

Similar Documents

Publication Publication Date Title
US8455435B2 (en) Remedies for ischemia
Brunner et al. Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow‐derived progenitor cells via the CXCR‐4/SDF‐1 axis
US8003096B2 (en) Means and methods for the recruitment and identification of stem cells
EP3011961B1 (de) 4f-benzoyl-tn14003 für die mobilisierung von hämotopoietischen vorläuferzellen im hinblick auf transplantation
KR101719339B1 (ko) 혈소판 수준을 증가시키는 펩타이드 요법
EP2549992B1 (de) Behandlungsverfahren mithilfe von stammzellenmobilisatoren
US20170119847A1 (en) Short beta-defensin-derived peptides
EP2007416B1 (de) Parathormon (pth) zur verwendung in der behandlung der ischämie
US20140088006A1 (en) Stem Cell Mobilization and Tissue Repair and Regeneration
CA2723270C (en) Unacylated ghrelin and analogs as therapeutic agents for vascular remodeling in diabetic patients and treatment of cardiovascular disease
KR101192136B1 (ko) 혈액 줄기 세포의 동원에 유효한 ｇ-ｃｓｆ와 ｐｌｇｆ의조합 약제
WO2017068583A1 (en) Methods and compositions for treating neurodegenerative diseases
US20230226150A1 (en) Methods for use of an angiocrine factor in treating a patient exposed to a myeloablative insult
KR101144687B1 (ko) Ｇ－ｃｓｆ 를 함유하는 선유아세포 동원제 및 창상 치료제
WO2001002003A1 (en) Method of treating psoriasis with il-15 antagonist
US20190374611A1 (en) Methods and pharmaceutical compositions for the treatment patients suffering from myeloproliferative disorders
WO2023230593A1 (en) Ptp1b inhibitors for treating lung injury
TAKAHASHI et al. FINAL ACCEPTED VERSION (H-00142-2006. R1)
Peled et al. Role of the CXCR4/CXCL12 Axis in Hematopoietic Stem Cell Trafficking

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20081009

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR MK RS

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20120827

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20170420

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 931375

Country of ref document: AT

Kind code of ref document: T

Effective date: 20171015

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602007052483

Country of ref document: DE

REG Reference to a national code

Ref country code: RO

Ref legal event code: EPE

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20171206

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Ref document number: 2007416

Country of ref document: PT

Date of ref document: 20180103

Kind code of ref document: T

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20171222

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: KELLER AND PARTNER PATENTANWAELTE AG, CH

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2654251

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20180212

REG Reference to a national code

Ref country code: EE

Ref legal event code: FG4A

Ref document number: E014710

Country of ref document: EE

Effective date: 20171222

REG Reference to a national code

Ref country code: SK

Ref legal event code: T3

Ref document number: E 26144

Country of ref document: SK

REG Reference to a national code

Ref country code: HU

Ref legal event code: AG4A

Ref document number: E035446

Country of ref document: HU

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180127

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20170403511

Country of ref document: GR

Effective date: 20180518

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 12

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602007052483

Country of ref document: DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20180628

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170927

REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 931375

Country of ref document: AT

Kind code of ref document: T

Effective date: 20170927

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180412

REG Reference to a national code

Ref country code: CH

Ref legal event code: PFA

Owner name: LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN, DE

Free format text: FORMER OWNER: LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN, DE

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: LT

Payment date: 20220328

Year of fee payment: 16

Ref country code: CH

Payment date: 20220322

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20220322

Year of fee payment: 16

Ref country code: SK

Payment date: 20220325

Year of fee payment: 16

Ref country code: RO

Payment date: 20220330

Year of fee payment: 16

Ref country code: PT

Payment date: 20220322

Year of fee payment: 16

Ref country code: PL

Payment date: 20220322

Year of fee payment: 16

Ref country code: IT

Payment date: 20220322

Year of fee payment: 16

Ref country code: FR

Payment date: 20220330

Year of fee payment: 16

Ref country code: EE

Payment date: 20220322

Year of fee payment: 16

Ref country code: CZ

Payment date: 20220324

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20220419

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20220422

Year of fee payment: 16

Ref country code: LU

Payment date: 20220412

Year of fee payment: 16

Ref country code: IE

Payment date: 20220413

Year of fee payment: 16

Ref country code: HU

Payment date: 20220404

Year of fee payment: 16

Ref country code: GB

Payment date: 20220422

Year of fee payment: 16

Ref country code: ES

Payment date: 20220502

Year of fee payment: 16

Ref country code: DK

Payment date: 20220422

Year of fee payment: 16

Ref country code: DE

Payment date: 20220419

Year of fee payment: 16

Ref country code: CY

Payment date: 20220405

Year of fee payment: 16

Ref country code: BG

Payment date: 20220331

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SI

Payment date: 20220322

Year of fee payment: 16

Ref country code: LV

Payment date: 20220421

Year of fee payment: 16

Ref country code: GR

Payment date: 20220503

Year of fee payment: 16

Ref country code: FI

Payment date: 20220427

Year of fee payment: 16

Ref country code: BE

Payment date: 20220421

Year of fee payment: 16

Ref country code: AT

Payment date: 20220407

Year of fee payment: 16

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602007052483

Country of ref document: DE

REG Reference to a national code

Ref country code: LT

Ref legal event code: MM4D

Effective date: 20230412

REG Reference to a national code

Ref country code: EE

Ref legal event code: MM4A

Ref document number: E014710

Country of ref document: EE

Effective date: 20230430

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

Effective date: 20230430

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: SK

Ref legal event code: MM4A

Ref document number: E 26144

Country of ref document: SK

Effective date: 20230412

Ref country code: NL

Ref legal event code: MM

Effective date: 20230501

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 931375

Country of ref document: AT

Kind code of ref document: T

Effective date: 20230412

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20230412

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20230430

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231110

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: SI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230413

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230413

Ref country code: RO

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231012

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230501

Ref country code: LV

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: LT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: HU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230413

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231110

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: EE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231103

Ref country code: CZ

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: CY

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

REG Reference to a national code

Ref country code: SI

Ref legal event code: KO00

Effective date: 20240116

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230412

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20240528